In this reporting period we have made significant progress in our studies. 1. Resolving the specific functional contributions of TRPC1 and Orai1 channels in polarized secretory epithelial cells. Earlier studies have shown that basolateraly localized TRPC1 is a major determinant of Ca2+ entry in acinar cells and thus required for sustained saliva flow. While Orai1 is localized near the apical region of the cell, its contribution to salivary gland function is not known. Here we have determined the individual contributions of TRPC1 and Orai1 in Ca2+-dependent activation of Kca channels, basolateral NKCC1, and apical TMEM16A, all of which are critical for fluid secretion. We report that report TRPC1 provides necessary Ca2+I in both apical and basolateral region of the cell to regulate these mechanisms while Orai1 has minimal direct contributions to regulation fluid secretion. Consistent with its basolateral localization, CCh-stimulated upregulation of NKCC1 was decreased >80% in acini from TRPC1-/- mice. Importantly, Ca2+ required for sustained activation of apically localized TMEM16A was also dependent on TRPC1-mediated Ca2+ entry. However, Orai1 is critical for fluid secretion since it is required for activation of TRPC1. Knockout of Orai1 within salivary glands of Oraifl/fl or TRPC1-/-/Orai1fl/fl mice by delivery of adCRE induced loss of fluid secretion and CCh-stimulated Ca2+ entry in acini to the same extent as in TRPC1-/- mice. Furthermore, overexpression of Orai1 and STIM1 in vivo in salivary glands of TRPC1-/- mice resulted in basolateral expression of Orai1 in acinar cells together with recovery of fluid secretion and Ca2+ entry. Thus the location, and possibly the expression level, of Orai1 in acinar cells determines its contribution to saliva flow. Together these findings suggest that TRPC1-mediated Ca2+ entry via the basolateral region is key to regulation of both apical and basal mechanisms involved in fluid secretion. 2. Mechanisms regulating plasma membrane recycling of TRPC1 channels: TRPC1 is the predominant channel contributing to SOCE in salivary gland cells. Previous studies revealed that TRPC1 is recruited to the plasma membrane upon Ca2+store depletion, resulting in enhancement of Ca2+entry. However, the mechanisms involved in the trafficking of TRPC1 to the cellular surface have not yet been identified. We show that endocytosis of TRPC1 is dependent on a mechanism that involves Arf6 and Rab5. Further, recycling back to the plasma membrane depends of Rab4. Together, our data suggest that Arf6, Rab5 and Rab4 play a critical, and specific, role in the endocytic recycling of TRPC1 and determine the level and activity of TRPC1 in the plasma membrane. Modulators of this mechanism can be suggested to have significant impact on salivary gland function. 3. STIM2 tunes receptor-stimulated Ca2+ signaling by escorting STIM1-Orai1 to the ER-PM junctional domains: We report that STIM2 tunes agonist-activation of SOCE by recruiting STIM1 to these microdomains at physiologically relevant intensities of agonist stimuli. Targeted deletion of STIM2 in mouse salivary glands diminished agonist-stimulated fluid secretion in vivo and SOCE activation in dispersed acinar cells. Interestingly, the effect of in vivo knockdown of STIM2 is particularly prominent at low agonist. To further investigate how STIM2 modulates Ca2+ signals induced by a range of agonist, we looked agonist-induced Ca2+ responses in single HEK293 cells. Loss of STIM2 decreased the agonist-sensitivity of SOCE activation and nuclear translocation of NFAT. Remarkably, deletion of the STIM2 polybasic domain impaired the recruitment of STIM1 and Orai1 into ER-PM junctional domains at low agonist. Together, our findings suggest that the major physiological role of STIM2 is to escort STIM1 into ER-PM junctional domains at low stimulus intensities when ER-Ca2+ stores are mildly depleted, thus increasing the agonist-sensitivity of Ca2+ signaling. 4. Recruitment of a cytoskeletal-regulatory complex with Orai1 and STIM1 during activation of SOCE: Little is known about other protein-protein interactions involving STIM1 (both in the resting state as well the changes as the activation process occurs). In this study, we identified 155 specific STIM1 binding partners using a shotgun proteomic approach using SILAC with a STIM1 immunoprecipitated complex from HSG cell lysates. Our analysis revealed several interesting changes in the STIM1 proteome upon stimulation with Tg (although, as expected, a majority of the binding partners were not affected by activation). As a result of the SILAC findings, our research has now focused on the role and interactions that cytoskeletal regulatory proteins appear to associate the SOCE core complex. For example proteins such as CD42, ARP2, N-WASP, septin, and zyxin interact with the SOCE proteins STIM1 and Orai-1. Knockdown of these results in decreases SOCE in the order CDC42>Septin>ARP2/3>N-WASP. We are presently trying to understand the interactions of these with cytoskeletal changes and assembly of Orai1-STIM1 channels. 5. Involvement of mitochondria in salivary gland damage in IR: Here we have explored the mechanism of IR-induced permanent damages of salivary gland cells by assessing mitochondria, as the primary cellular target for IR. Mitochondria reactive oxygen species (mROS) in HSG cells were elevated after IR. The elevation was attenuated by maintaining cells in Ca2+-free medium during IR, or by treatment with Tempol. Further, mitochondrial membrane potential (m&#936;) was decreased (depolarization) and mitochondrial Ca2+ (mCa2+) was significantly increased in irradiated cells. Both of these were blocked by inhibition of the mitochondrial Ca2+ uniporter (MCU) with Ru360, treating cells with siMCU, or removing external Ca2+ during IR. More importantly cells treated with either Ru360 or siMCU showed greater viability than control cell 24 and 96 hours after irradiation. Our data suggest that IR causes pathophysiological changes in mitochondria that lead to loss of cell function and cell death. 6. Characterization of Ca2+ signaling in salivary gland biopsies from SS patients: Sjgrens syndrome (SS) is an autoimmune disease associated with lymphocytic infiltration and reduced fluid secretion in salivary glands that results in xerostomia. Intriguingly, there is substantial loss of gland function with minimal infiltration or tissue damage. Here we have examined single acinar cell function in minor salivary gland biopsies from SS patients and healthy volunteers. Glands from SS patients with low inflammation showed acinar destruction within the small areas of infiltration; while a large part of the tissue was relatively intact. Importantly, acinar cells in these areas of the glands displayed significant attenuation of Carbachol (Cch) -stimulated intracellular Ca2+ release and Ca2+ entry; as compared to those from healthy volunteers. Consistent with this, agonist-stimulated volume decrease, representing fluid secretion was also lower in these acini. In contrast to the poor correlation between inflammation and saliva flow, there was significant correlation between CCh-stimulated cellular responses and saliva secretion within the patient population. IP3R2 & IP3R3, but not AQP5 or STIM1, were decreased in the intact areas of the gland which can account for the disruption in Ca2+ signaling and consequently fluid secretion. Together, our findings reveal that IP3R deficit in acinar cells underlies the loss of fluid secretion in SS patients who display significant xerostomia.